Cholera: Death by diarrhoea

FACTFILE : Death by diarrhoea

• Cholera is an infectious disease caused by the bacterium , which affects the transport of water in the small intestine. The bacterium secretes a toxin, cholera toxin (CT), which causes severe fluid loss from the body into the digestive tract, which leads to dehydration and ultimately death by diarrhoea.

• V. cholerae has caused seven worldwide disease outbreaks or since 1817, killing millions of people and infecting millions more, including four epidemics in the UK accounting for approximately 55,000 deaths.

• Southeast Asia witnessed the first cholera epidemic in 1817, though the condition of ‘dehydrating diarrhoea’ was mentioned around the time of Hippocrates (460–377 BC). Today, cholera is prevalent in Central and South America, Africa and Asia, though not in the UK as it is usually confined to countries with poor sanitation infrastructure. Worldwide 100,000–300,000 cases of the disease are reported each year with more than 94% of cases in Africa. However, this is likely to be a massive underestimate as many countries in the Indian subcontinent and Southeast Asia do not report cholera incidence; for example, no cholera cases have been reported in Bangladesh, although estimates suggest there may be as many as 1 million cases per year. Realistic worldwide estimates suggest 120,000 annual deaths with 3–5 million people suffering from the disease. Coloured transmission electron micrograph of a Vibrio cholerae bacterium.

2 Factfile: Cholera | www.microbiologysociety.org Cholera: a history Prior to 1850 it was thought that cholera was caused by breathing in bad air – ‘miasma’ – and that protection was offered by strong smelling substances such as herbs and camphor. , a prominent London physician was heralded as the ‘Father of Cholera’ when he questioned the miasma theory after treating a number of cholera patients and not succumbing to the condition himself. Snow argued that disease must have entered the body through the mouth via infected food or drink and not via the lungs, after observing that patients became ill with symptoms originating in the gut such as vomiting and stomach ache. He hypothesised that a ‘poison’ was ingested which reproduced in the digestive tract, and in 1849 he published his ideas, at his own expense, in a pamphlet called On the Modes of Communication of Cholera. Snow was able to prove his theory in 1854 when he carefully mapped the John Snow’s cholera map of Soho, London, showing deaths (black lines) due to cholera during the epidemic of 1854. incidence of cholera during the London source of infection; however, through describing the causative agent of 1853–1854 outbreak. Believing that data gathering and statistical analyses cholera, and suggesting treatment the source was a water pump in Broad he suggested that the water was the options. However, the preferred theory Street, Soho he examined water from reservoir of infection and the mode of in Italy at that time was that of miasma the pump under the microscope and transmission. His recommendations of and as such Pacini’s work went described the occurrence of “white improved hygiene and boiling of drinking unnoticed. flocculent particles”. Convinced that water mostly went unheeded by those A German bacteriologist, Robert these particles were the source of the who did not support his claims as the Koch, suggested in 1882 that cholera disease he persuaded the authorities to ‘germ’ responsible for cholera had not was caused by a bacterium and that remove the pump handle; at this time yet been observed microscopically. this bacterium secreted a toxin which the number of cholera cases locally was In the same year that Snow caused rapid water loss. Koch observed already in decline, though it is thought made his observations an Italian faecal samples and identified a comma- Coloured transmission electron that Snow’s conclusions provided the anatomist, Fillipo Pacini, published a shaped bacillus which he called Vibrio micrograph of a Vibrio cholerae basis for further preventative measures. paper entitled Microscopical observations cholerae after its vibrating wiggles. bacterium. John Snow never identified the bacterial and pathological deductions on cholera Having already made his mark on

Factfile: Cholera | www.microbiologysociety.org 3 bacteriology with his work on anthrax and tuberculosis, and his postulates (see below), which generate a formal proof that a certain micro-organism causes a disease, his ideas and observations on cholera were well received. Pacini was recognised for his work posthumously as the organism was officially named Vibrio cholerae Pacini 1854.

What causes cholera? Organism Cholera is caused by the curved, rod-shaped bacterium Vibrio cholerae. Artwork of the human large intestine There are more than 100 species or surrounded by some of the micro-organisms O serotypes of Vibrio, only a few of that can cause diarrhoea.

which are pathogenic to humans. Koch’s postulates They are free-living bacteria, but Robert Koch developed a set of Postulate 1: closely associated with the presence postulates for proving that a specific The suspected pathogenic micro- of zooplankton and crustaceans, which micro-organism caused a specific organism should be present in all are commonly found in brackish waters. disease. cases of the disease and absent from Vibrio cells are motile, propelled by healthy individuals [this postulate is a single polar flagellum and, as they often disregarded as, in the case of normally live in neutral or alkaline cholera for example, it is possible to conditions they are sensitive to be a carrier and not show symptoms acidic pH. The bacteria are tiny; of the disease (asymptomatic)]. about one five hundredth of a millimetre long which means Postulate 2: twenty or more would fit across The suspected micro-organism the diameter of a human hair. should be grown in pure culture (note the correct media must be selected Getting there… for successful culture). Cholera infection results from the ingestion of contaminated food or water. Postulate 3: To survive the journey through the Cells from a pure culture of the digestive tract the bacterium has to suspected micro-organism should be well adapted. cause disease in a healthy animal. Passage through the digestive tract Postulate 4: Entry to the body: V. cholerae enters The micro-organism should be the body and makes its way through re-isolated and shown to be the same the digestive system. Approximately The German bacteriologist Robert Koch, 1843–1910. as the original. two-thirds survive the acidic conditions of the stomach; survivors conserve

4 Factfile: Cholera | www.microbiologysociety.org to the body’s defence mechanisms. excreting the bacteria in their faeces, ‘Cholera Dolores’ To conserve energy, on reaching usually for two weeks but occasionally Asymptomatic cholera infection the intestine wall the bacteria stop for several years as in the case of is unlikely to last more than two flagellum production, as propulsion ‘Cholera Dolores‘. weeks; however, long-term carriers is no longer required, and energy is have been reported, though they refocused on the production of hair-like Different types ofV. cholerae infection are extremely rare. During the appendages called fimbriae or pili which Over 100 serogroups (organisms Philippine epidemic of 1962, Dolores, are formed on the bacterial cell surface. grouped on the basis of their cell a housewife, suffered a mild attack These structures are made of protein surface antigens) of V. cholerae exist; of cholera while pregnant with her and allow the bacteria to attach to the however, only two cause epidemic sixth child, and was hospitalised lining of the intestine. cholera. They are serogroup O1 and though not treated with . O139. Serogroup O1 can be classified It is thought that she contracted Growth: For symptoms to persist, the further based on phenotype into cholera from eating contaminated bacteria must continue to multiply in the El Tor and Classical; these are seafood which her husband brought intestine. referred to as their biotypes. El Tor from nearby Bacolod City. Several is the infective agent responsible for members of Dolores’ family showed Colonisation and Invasion: When the current ; Classical has symptoms of cholera, though none they reach the small intestine, the not been identified since the mid-1990s. needed hospitalisation. Dolores bacteria must ‘hold on’ and resist the V. cholerae O1 biotypes can be further intermittently excreted the bacteria normal transit motions of this region. classified into serotypes based on the so had the potential to cause further They do this using their fimbriae or pili. results of a serum agglutination test; infection although this was never Once attached the bacteria invade the there are three O1 serotypes, Inaba, observed in her locality. This is host cell. Production of CT is the final Ogawa and Hikojima. V. cholerae possibly due to the fact that she stage in pathogenesis. O139, a relatively new serogroup, lived in an area where cholera was has not reached epidemic potential endemic. Little more is known of Causing trouble… so far, though its occurrence is being this case except that the V. cholerae The period between ingestion and a closely monitored as its pattern of was resident in her biliary tract; it is patient showing symptoms (incubation infection and capacity to survive in unknown what factors contributed period) is usually very short and can water suggest that it may be both to Dolores being a carrier for such be as little as 2 hours, although it can more infectious and virulent than a long period of time. In 1973, take up to 5 days. Most people (75%) V. cholerae O1. Dolores’ carrier state resolved infected with V. cholerae don’t have any It is worth noting that the gene for spontaneously. symptoms (asymptomatic); however CT is carried on a bacteriophage (a virus they remain carriers of the disease, which infects bacteria) inside V. cholerae. energy until they enter the small Classification of V. cholerae infection intestine where they begin production of their flagella. The flagellum, which is Bacteria V. cholerae a long tail-like structure, propels each Vibrio cell forward through the mucus Serogroup O1 O139 layer to the intestinal wall. V. cholerae also produces enzymes which digest the layers of mucus, which helps with Biotype El Tor Classical access to the epithelial cells lining the intestine, as well as detachment Serotype Inaba Hikojima Ogawa Inaba Hikojima Ogawa from the cells which are being lost due

Factfile: Cholera | www.microbiologysociety.org 5 a lack of consciousness, confusion and occasionally fever. It is this rapid dehydration – as much as 1 litre of fluid can be lost every hour – which can prove fatal within 24 hours of developing the disease. In children, the rapid loss of fluid can lead to severe salt imbalance, which can cause convulsions and cardiac arrest in addition to the symptoms described. The fatality rate in untreated cases can be as high as 30–50%. If patients are given rehydration therapy, then the death rate is below 1%.

Laboratory methods for diagnosis of V. cholerae Many diseases cause diarrhoea; however, the production of frequent watery stools requires faster treatment due to the high risk of dehydration. Looking at a stool sample under the microscope can help the medical practitioner make a diagnosis. However, this is of little use to patients Bacteriologist holding a Petri dish containing colonies of Vibrio cholerae bacteria. as those with any diarrhoeal disease receive the same treatment, i.e. fluid So, in theory if this virus were more pathogens, such as viruses. However, and electrolyte (salts and sugars) mobile it might be possible for a new all of these people carry V. cholerae, replacement. serotype to emerge again in the future. excrete it and can spread it to others. Conventional culture methods Alternatively, V. cholerae species could Severe cholera (cholera gravis), which are the ‘gold standard’ for diagnosis swap genes involved in the production accounts for fewer than 10% of cases, is of V. cholerae. Stool samples or rectal of O antigens as has happened for O139 characterised by: swabs on TCBS (thiosulphate citrate and O37. These serotypes have a genetic • rapid onset of violent watery bile salts) agar (V. cholerae colonies fingerprint resembling O1 serotype diarrhoea – the diarrhoea is ‘straw appear yellow as they ferment glucose). strains, but in both cases the O1 antigen coloured’ with flecks of mucus and Suspected colonies are selected for gene cluster has been replaced by a is often described as resembling further analysis with biochemical non-O1 gene cluster. rice water and serological tests such as slide • vomiting agglutination. Symptoms In cholera endemic areas, where • extensive dehydration Most people with cholera are laboratory facilities are often limited, a asymptomatic and, of those that • leg cramps rapid immunochromatographic dipstick do develop symptoms, 80% have As fluid is lost the blood thickens and test can be used to diagnose cholera. mild diarrhoea which is difficult to the skin becomes blue-grey in colour; It is very simple to use; the dipstick is distinguish from that caused by other patients also begin to suffer lethargy, dipped into a stool sample. If two red

6 Factfile: Cholera | www.microbiologysociety.org Two girls washing dishes in muddy river water on the Niger River in Mali. Using unclean water can lead to infections from parasites, bacteria or other microbes. lines appear on the dipstick, then the is not often used in treatment, this contaminated with infected excrement. patient has cholera, if only one red line information helps to prevent further The bacteria can also be spread to food appears, the test is negative. It takes outbreaks. if infected people don’t wash their hands between 2 and 15 minutes for the test thoroughly after going to the toilet and to develop. Transmission of cholera before food preparation. Infection is Retrospective diagnosis can be Cholera infection requires a large often noted after a funeral of a cholera performed by analysis of the blood for dose of bacteria and is transmitted victim where contamination occurs antibodies against V. cholerae and CT. by the faecal–oral route, for example as a result of poor hygiene in food Although identification of V. cholerae by drinking from a water supply preparation.

Drinking water supplies being distributed during a cholera epidemic in 19th century Hamburg, Germany.

Factfile: Cholera | www.microbiologysociety.org 7 The environment can also be a reservoir of infection as the bacteria are present in brackish waters, often in association with aquatic organisms at a low level, and sometimes in an unculturable state. Shellfish living in contaminated water can transmit cholera too. They are filter feeders and, as they strain the water for food, the bacteria become concentrated inside them. Anyone consuming shellfish that are not properly cooked can become ill.

Treatment Treatment of the symptoms of cholera focuses on fluid replacement as water Patients being treated in a cholera treatment centre, run by Médecins Sans Frontières, in Port-au-Prince, Haiti. alone is not easily absorbed by the body. The World Health Organization (WHO) recommends clean water and rehydration salts to restore electrolyte balance. (ORT) employs rehydration salts, which are readily available in pharmacies; these are often in short supply in areas of high infection rate. ORT has been revolutionary in cholera treatment and, if properly used, it hugely reduces both the need for hospitalisation and mortality. Some cases may be treated with antibiotics although generally the individual’s immune system is left to destroy the pathogen. In extreme cases, Hartmann’s solution (a solution of sodium, chloride, lactate, potassium and calcium ions, which is isotonic with blood) is administered by intravenous injection to replace body fluid and mineral salts.

Prevention Good sanitation, clean drinking water Hartmann’s solution is used to replenish body fluid and restore normal salt concentrations for patients who have and improved hygiene practices, such become severely dehydrated after an illness. as washing hands after visiting the

8 Factfile: Cholera | www.microbiologysociety.org Food Drink

Cook all foods thoroughly Use bottled, boiled, or chemically treated water to wash dishes, clean teeth and prepare food

Raw fruit and vegetables should Only drink bottled water be avoided unless you thoroughly that has the seal still intact wash and peel them yourself

Ice cream from doubtful sources Avoid ice cubes in drinks may also be contaminated therefore should be avoided

Food must be properly prepared Boil unpasteurised milk and still hot when it is served before you drink it

Be careful when eating food from street stalls

Avoid salads as they may have been washed in contaminated water

Avoid raw fish and shellfish Clean and dirty water in glasses. toilet and before preparing food, are acts as a first-line of defence against example immunity provided against V. all strategies that need to be followed infection and kills many V. cholerae cholerae O1 does not protect from O139. to prevent the outbreak of cholera. cells before they travel to the intestine. The WHO recommends vaccination for The table above shows some tips for Another possibility for physiological people occupying slums or refugee travellers on how to avoid infection, variation among infected individuals camps due to high density populations from food and drink, when visiting an may be the availability of surface and increased risk of disease spread. area where cholera is present. A simple receptors for CT on the host cell surface, In 1894, soon after Koch’s rule is: boil it, cook it, wash and peel it or though this has not been proven. identification of cholera, an injectable, forget it. whole-cell, killed vaccine was developed Vaccines and Immunity that induced 48% protection for three Physiological differences Oral vaccines show long-lasting months against V. cholerae serotypes affecting infection protection against cholera with few Inaba and Ogawa. The vaccine, initially Cholera infection can be spectral in side-effects, although they provide trialled in India, was never endorsed by that symptoms can vary with dose, insufficient protection for children WHO, and is no longer available. serogroup, biotype, and serotype, though under the age of 2. Vaccines, however, Protective immunity in those physiological variation between people are only advised for preventative use exposed to cholera is induced almost can also play a part. It is thought that rather than as a method for controlling exclusively by antibodies produced those who have reduced or non-existent outbreaks and should be used in tandem in the intestine. These antibodies stomach acid due to disease or ailment with standard prevention and control prevent bacterial colonisation and are more susceptible to cholera. In a measures. Also immunity, natural or multiplication, and they inactivate the CT. healthy individual, the stomach acid artificial, is serogroup-specific; for Immunoglobulins IgA, IgG and IgM have

Factfile: Cholera | www.microbiologysociety.org 9 all been detected, although IgA are the In areas where cholera is endemic Future most important. The antibodies prevent such as Bangladesh, infection rates are Current research into cholera is the CT from binding with the receptors low among adults when compared to focused on understanding the CT on the cell surface. Natural immunity the children in the same area, whereas itself. Another area of anti-diarrhoeal is provided by IgM followed by a switch in areas where new epidemics arise research is development of a pill to IgG. There is a 3-year period post rates are higher in the adult population. that will prevent the diarrhoea post- infection where patients remain immune This discrepancy illustrates a resistance infection. Two drugs, chlorpromazine from cholera as a result of acquired, linked to the presence of circulating and nicotinic acid, have been shown to natural immunity. vibriocidal antibodies to V. cholerae. be effective in animal models; though Effective oral vaccines available against cholera

WC/rBS (Dukoral) to assist with the containment of cholera been shown in an effective trial, though This is an oral killed, whole-cell outbreaks and epidemics in susceptible retrospective studies have shown V. cholerae O1 vaccine with the populations. The stockpile has already protection in an ongoing outbreak in recombinant B (binding) subunit of been used to great effect. Micronesia, a group of islands in the CT. Trials have shown high levels of Western Pacific. Orochol was the only protection (85–90%) over a period of CVD 103-HgR vaccine available as a single dose, 6 months for 2 doses of the vaccine (Vaxchora™, previously Orochol) which, due to administration logistics, in all age groups, though protection This vaccine consists of an attenuated, is more viable for pre-emptive and declines after 6 months in young live, genetically modified V. cholerae O1 long-term outbreak control in ongoing children and remains at about 60% in Inaba strain that has been engineered field conditions according to WHO. older children and adults after 2 years. to produce part of the CT. It confers high However, due to a lack of evidence Protection is provided against V. cholerae protection (95%) against O1 (Classical for its effectiveness, the vaccine was O1 serotypes Inaba and Ogawa, and and El Tor) in virgin volunteers, i.e. those withdrawn in 2004, now redeveloped biotypes Classical and El Tor. Dukoral not previously exposed to the bacterium. as Vaxchora™, which is in the advanced also provides short-term protection Protection in endemic regions has not stages of application for a US licence. against Escherichia coli enterotoxin, which is of added benefit to travellers. This vaccine is licenced in Europe.

Bivalent variants of WC (Euvichol, m-ORCVax and Sanhancol) These are cheaper versions of the oral killed WC/rBS and are licenced in the Republic of Korea, Vietnam and India respectively. These vaccines lack the B subunit of CT and contain twice as many killed V. cholerae cells, including cells of serotype O139. Two years after vaccination, the efficacy is approximately 66% in all age groups including infants. The WHO has built a stockpile of oral killed Cholera vaccines Child being given an oral vaccine in Afghanistan.

10 Factfile: Cholera | www.microbiologysociety.org the mechanism is yet to be understood. Research into development of a live attenuated Cholera vaccine continues, and it is currently in clinical trials in Australia and the US. Following promising field trials of the bivalent oral killed Cholera vaccine in Bangladesh, the introduction of this vaccine in the national vaccination programme is being considered.

Education In order to control and ultimately stop the spread of a cholera outbreak, community education is paramount. Many of the basic hygiene messages, while perhaps simple to relate, are often difficult to implement due to cost. Therefore, alternative solutions are required to limit transmission, for example the WHO suggests the addition of lime juice to food and water to inactivate V. cholerae. Ideally in communities most affected by cholera, awareness campaigns continue throughout the year, increasing in frequency as the cholera season approaches. Clear information is presented with messages adapted for culture, social and economic circumstances, and the information is delivered graphically, by radio broadcast or as talks in areas where people are waiting or congregated. Coloured transmission electron micrograph of a Vibrio cholerae bacterium.

Sources of further information Centers for Disease Control www.cdc.gov/nczved/divisions/dfbmd/diseases/cholera European Centre for Disease Prevention and Control www.ecdc.europa.eu/en/healthtopics/cholera Net Doctor www.netdoctor.co.uk/travel/diseases/cholera.htm BBC www.bbc.co.uk/scotland/education/int/geog/health/health/cholera/causes World Health Organization www.who.int/cholera/vaccines/double/en Textbook of Bacteriology www.textbookofbacteriology.net/cholera.html

Factfile: Cholera | www.microbiologysociety.org 11 Factfile: Cholera: Death by diarrhoea First edition 2009; revised 2016 Microbiology Society Acknowledgements The Microbiology Society is a membership organisation Thanks are due to Professor Charles Penn (University for scientists who work in all areas of microbiology. of Birmingham) and Dr Sjoerd Rijpkema (NIBSC) for their helpful comments on this text. Every care has It is the largest learned microbiological society in been taken to ensure that the information is correct, Europe with a worldwide membership based in but the author will be pleased to learn of any errors universities, industry, hospitals, research institutes and that have remained undetected. schools. The Society publishes key academic journals, Picture credits organises international scientific conferences and Front/back cover and p. 2, AMI Images/SPL*; provides an international forum for communication p. 3, British Library/SPL; p. 4 top, John Bavosi/SPL; bottom, National Library of Medicine/SPL; p. 6, among microbiologists and supports their professional Mauro Fermariello/SPL; p. 7 top, Peter Menzel/SPL; development. The Society promotes the understanding bottom, SPL; p. 8 top, Julie Dermansky/SPL; p. 8 bottom, Antonia Reeve/SPL; p. 9, Gustoimages/SPL; of microbiology to a diverse range of stakeholders, p. 10, Ton Koene/Visuals Unlimited, Inc./SPL; p. 11, including policymakers, students, teachers, journalists Dr Kari Lounatmaa/SPL.*SPL, Science Photo Library and the wider public, through a comprehensive Copyright framework of communication activities and resources. Cholera: Death by diarrhoea is copyright. Microbiology The Society produces and distributes a wide range of Society asserts its moral right to be identified under Section 77 of the Design, Patents and Copyright Act, resources including videos to support microbiology UK (1988). teaching in schools and colleges. Educational use: electronic or paper copies of the resources or individual pages from it may be made for classroom use and bona fide educational resources, provided that the copies are distributed free of charge For more information, see www.microbiologyonline.org.uk or contact the or at the cost of reproduction and Microbiology Society Education Department, Microbiology Society, Charles Darwin House, 12 Roger is credited and identified as copyright holder. Street, London WC1N 2JU, UK ([email protected]).

© 2016 Microbiology Society

Front cover and right Coloured scanning electron micrograph of a Vibrio cholerae bacterium.